The long-term objective of this proposal is to improve the therapy of human glioma by better understanding the determinants of resistance to TRAIL. TRAIL is an attractive therapeutic molecule because it induces apoptosis in tumor cells, but not in normal cells. Even in TRAIL sensitive glioblastoma multiforme (GBM) cell lines, however, TRAIL resistance is common. Although TRAIL resistance can be controlled by many factors, we have found that over-expression of FLIPs, a caspase-8 inhibitor that blocks activation of the extrinsic apoptotic cascade, plays a key role in the high level TRAIL resistance common in primary GBM. FLIPs over- expression is not associated with increased FLIPs mRNA levels, but with increased association of FLIPs mRNA with translating polyribosomes. FLIPs mRNA translation is in turn influenced by two arms of the Ras pathway, both activated in GBM: the Ras-Ral arm of the pathway blocks association of FLIPs mRNA with polysomes, downregulates FLIPs protein, and sensitizes GBM cells to TRAIL, while the Ras-Akt arm of the pathway enhances FLIPs mRNA polysomal association, upregulates FLIPs protein, and protects GBM cells from TRAIL. The Ral and Raf arms of the Ras pathway, however, also appear to suppress FLIPs RNA levels, suggesting that transcriptional control of FLIPs may also be important. We do not know how the Ral Akt, and Raf pathways control FLIPs mRNA levels and translation, how the FLIPs mRNA is differentially targeted for translation regulation, if the FLIPs mRNA is part of a larger group of apoptosis-related mRNAs targeted for translational control, whether the regulation noted in GBM cell lines also occurs in primary gliomas, or how to best manipulate the pathways for therapeutic benefit. Based on our preliminary studies, however, we hypothesize that Ras-related translational and transcription regulation of FLIPs controls TRAIL sensitivity in GBM. This hypothesis will be tested in the following specific aims. 1: To define the connections between Ral/Akt, regulators of translation, FLIPs levels, and TRAIL sensitivity in GBM cells. 2: To identify FLIPs mRNA sequences responsible for Ras-dependent FLIPs translational regulation. 3: To determine if FLIPs is part of a larger group of translationally regulated molecules that contribute to the control TRAIL-induced apoptosis. 4: To define the connections between Raf/Ral, downregulation of FLIPs RNA, and TRAIL sensitivity in GBM. 5: To define TRAIL sensitivity in a panel of short term cultures of primary human low grade gliomas (LGG), anaplastic astrocytoma (AA), and GBM, and to determine if levels/activity of Ras pathways and/or FLIPs correlate with TRAIL sensitivity. 6: To determine if modulation of translation control can sensitize primary human GBM to TRAIL. This work has relevance to public health in that it will provide the ways to identify TRAIL-sensitive brain tumors and also to make TRAIL-resistant tumors sensitive to TRAIL-induced killing.